Improving the delivery of drugs and other agents to target cells and tissues has been the focus of considerable research for many years. Though many attempts have been made to develop effective methods for importing biologically active molecules into cells, both in vivo and in vitro, none has proved to be entirely satisfactory. Optimizing the association of the inhibitory drug with its intracellular target, while minimizing intercellular redistribution of the drug, e.g., to neighboring cells, is often difficult or inefficient.
Most agents currently administered to a patient parenterally are not targeted, resulting in systemic delivery of the agent to cells and tissues of the body where it is unnecessary, and often undesirable. This may result in adverse drug side effects, and often limits the dose of a drug (e.g., glucocorticoids and other anti-inflammatory drugs) that can be administered. By comparison, although oral administration of drugs is generally recognized as a convenient and economical method of administration, oral administration can result in either (a) uptake of the drug through the cellular and tissue barriers, e.g., blood/brain, epithelial, cell membrane, resulting in undesirable systemic distribution, or (b) temporary residence of the drug within the gastrointestinal tract. Accordingly, a major goal has been to develop methods for specifically targeting agents to cells and tissues. Benefits of such treatment includes avoiding the general physiological effects of inappropriate delivery of such agents to other cells and tissues, such as uninfected cells.
Many of the current treatment regimes for cell proliferation diseases such as psoriasis and cancer utilize compounds which inhibit DNA synthesis. Such compounds are toxic to cells generally but their toxic effect on rapidly dividing cells such as tumor cells can be beneficial. Alternative approaches to anti-proliferative agents which act by mechanisms other than the inhibition of DNA synthesis have the potential to display enhanced selectivity of action.
In recent years it has been discovered that a cell may become cancerous by virtue of the transformation of a portion of its DNA into an oncogene i.e. a gene that, on activation, leads to the formation of malignant tumor cells (Bradshaw, Mutagenesis 1986, 1, 91). Several such oncogenes give rise to the production of peptides which are receptors for growth factors. The growth factor receptor complex subsequently leads to an increase in cell proliferation. It is known, for example, that several oncogenes encode tyrosine kinase enzymes and that certain growth factor receptors are also tyrosine kinase enzymes (Yarden et al., Ann. Rev. Biochem., 1988, 57, 443; Larsen et al., Ann. Reports in Med. Chem. 1989, Chpt. 13).
Receptor tyrosine kinases are important in the transmission of biochemical signals that initiate cell replication. They are large enzymes that span the cell membrane and possess an extracellular binding domain for growth factors such as epidermal growth factor (EGF), and an intracellular portion that functions as a kinase to phosphorylate tyrosine amino acids in proteins and hence influence cell proliferation. Various classes of receptor tyrosine kinases are known (Wilks, Advances in Cancer Research, 1993, 60, 43-73) based on families of growth factors thath bind to different receptor tyrosine kinases. The classification includes Class I receptor tyrosine kinases comprising the EGF family of receptor tyrosine kinases such as the EGF, TGFα, NEU, erbB, Xmrk, HER and let23 receptors, Class II receptor tyrosine kinases comprising the insulin family of receptor tyrosine kinases such as the insulin, IGFI and insulin-related receptor (IRR) receptors and Class III receptor tyrosine kinases comprising the platelet-derived growth factor (PDGF) family of receptor tyrosine kinases such as the PDGFα, PDGFβ and colony-stimulating factor 1 (CSF1) receptors.
It is known that Class I kinases such as the EGF family of receptor tyrosine kinases are frequently present in common human cancers such as breast cancer (Sainsbury et al., Brit. J. Cancer, 1988, 58, 458; Guerin et al., Oncogene Res., 1988, 3, 21 and Klijn et al., Breast Cancer Res. Treat., 1994, 29, 73), non-small cell lung cancers (NSCLCs) including adenocarcinomas (Cerny et al., Brit. J. Cancer, 1986, 54, 265; Reubi et al., Int. J. Cancer, 1990, 45, 269; and Rusch et al., Cancer Research, 1993, 53, 2379) and squamous cell cancer of the lung (Hendler et al., Cancer Cells, 1989, 7, 347), bladder cancer (Neal et al., Lancet, 1985, 366), oesophageal cancer (Mukaida et al., Cancer, 1991, 68, 142), gastrointestinal cancer such as colon, rectal or stomach cancer (Bolen et al., Oncogene Res., 1987, 1, 149), cancer of the prostate (Visakorpi et al., Histochem. J., 1992, 24, 481), leukaemia (Konaka et al., Cell, 1984, 37, 1035) and ovarian, bronchial or pancreatic cancer (European Patent Specification No. 0400586). As further human tumor tissues are tested for the EGF family of receptor tyrosine kinases, it is expected that their widespread prevalence will be established in further cancers such as thyroid and uterine cancer. It is also known that EGF type tyrosine kinase activity is rarely detected in normal cells, whereas it is more frequently detectable in malignant cells (Hunter, Cell, 1987, 50, 823). It has been shown more recently (W. J. Gullick, Brit. Med. Bull., 1991, 47, 87) that EGF receptors that possess tyrosine kinase activity are overexpressed in many human cancers such as brain, lung squamous cell, bladder, gastric, breast, head and neck, oesophageal, gynaecological and thyroid tumors.
Accordingly, an inhibitor of receptor tyrosine kinases should be of value as a selective inhibitor of the growth of mammalian cancer cells (Yaish et al. Science, 1988, 242, 933). Support for this view is provided by the demonstration that erbstatin, an EGF receptor tyrosine kinase inhibitor, specifically attenuates the growth in athymic nude mice of a transplanted human mammary carcinoma that expresses EGF receptor tyrosine kinase but is without effect on the growth of another carcinoma that does not express EGF receptor tyrosine kinase (Toi et al., Eur. J. Cancer Clin. Oncol., 1990, 26, 722.) Various derivatives of styrene are also stated to possess tyrosine kinase inhibitory properties (European Patent Application Nos. 0 211 363, 0 304 493 and 0 322 738) and to be of use as anti-tumor agents. The in vivo inhibitory effect of two such styrene derivatives that are EGF receptor tyrosine kinase inhibitors has been demonstrated against the growth of human squamous cell carcinoma inoculated into nude mice (Yoneda et al., Cancer Research, 1991, 51, 4430). Various known tyrosine kinase inhibitors are disclosed in a more recent review by T. R. Burke Jr. (Drugs of the Future, 1992, 17, 119).
Kinase inhibitors have valuable pharmacological properties and can be used, for example, as anti-tumoral drugs and as drugs against atherosclerosis. The phosphorylation of proteins has long been known as an important step in the differentiation and proliferation of cells. Phosphorylation is catalyzed by protein kinases that are divided into serine/threonine kinases and tyrosine kinases. The serine/threonine kinases include protein kinase C and the tyrosine kinases include PDGF (platelet-derived growth factor)-receptor tyrosine kinase and Bcr-Abl kinase.
Kinase inhibitors inhibit cellular kinases that are involved in disease states, for example, Bcr-Abl. Chronic myelogenous Leukemia (CML) is a hematological stem cell disorder associated with a specific chromosomal translocation known as the Philadelphia chromosome that is detected in 95% of patients with CML and 20% with acute lymphocytic leukemia (ALL). The molecular consequences of the translocation is the fusion of the abl protooncogene to the bcr gene resulting in the production of an activated from of Abl tyrosine protein kinase. The Bcr-Abl protein is capable of inducing leukemias in mice, thus implicating the protein as the cause of these diseases. As the tyrosine kinase activity of the Bcr-Abl protein is essential to its transforming ability, an inhibitor would be useful therapy for these disorders.
In addition, kinase inhibitors prevent the development of resistance (multi-drug resistance) in cancer treatment with other chemotherapeutic drugs or remove existing resistance to other chemotherapeutic drugs.
Two processes, the de novo formation of vessels from differentiating endothelial cells or angioblasts in the developing embryo (vasculogenesis) and the growth of new capillary vessels from existing blood vessels (angiogenesis), are involved in the development of the vascular systems of animal organs and tissues. Transient phases of new vessel formation (neovascularization) also occur in the adult body, for example, during the menstrual cycle, pregnancy or wound healing. On the other hand, a number of diseases are known to be associated with deregulated angiogenesis, for example, retinopathies, psoriasis, hemangioblastoma, hemangioma, and neoplastic diseases (solid tumors). The complex processes of vasculogenesis and angiogenesis have been found to involve a whole range of molecules, especially angiogenic growth factors and their endothelial receptors, as well as cell adhesion molecules.
Recent findings show that at the center of the network regulating the growth and differentiation of the vascular system and its components, both during embryonic development and normal growth and in a wide number of pathological anomalies and diseases, lies the angiogenic factor known as vascular endothelial growth factor (VEGF), along with its cellular receptors (see Breier, G., et al., Trends in Cell Biology 6, 454-6 (1996) and the references cited therein).
VEGF is a dimeric, disulfide-linked 46-kDa glycoprotein and is related to platelet-derived growth factor (PDGF). It is produced by normal cell lines and tumor cell lines, is an endothelial cell-specific mitogen, shows angiogenic activity in in vivo test systems (e.g. rabbit cornea), is chemotactic for endothelial cells and monocytes, and induces plasminogen activators in endothelial cells, which are then involved in the proteolytic degradation of extracellular matrix during the formation of capillaries. A number of isoforms of VEGF are known that show comparable biological activity, but differ in the type of cells that secrete them and in their heparin-binding capacity. In addition, there are other members of the VEGF family, such as placenta growth factor (PLGF) and VEGF-C.
VEGF receptors are transmembranous receptor tyrosine kinases. They are characterized by an extracellular domain with seven immunoglobulin-like domains and an intracellular tyrosine kinase domain. Various types of VEGF receptor are known, e.g. VEGFR-1, VEGFR-2, and VEGFR-3.
A large number of human tumors, especially gliomas and carcinomas, express high levels of VEGF and its receptors. This has led to the hypothesis that the VEGF released by tumor cells could stimulate the growth of blood capillaries and the proliferation of tumor endothelium in a paracrine manner and thus, through the improved blood supply, accelerate tumor growth. Increased VEGF expression could explain the occurrence of cerebral edema in patients with glioma. Direct evidence of the role of VEGF as a tumor angiogenesis factor in vivo has been obtained from studies in which VEGF expression or VEGF activity was inhibited. This was achieved with antibodies that inhibit VEGF activity, with dominant-negative VEGFR-2 mutants that inhibited signal transduction, or with the use of antisense-VEGF RNA techniques. All approaches led to a reduction in the growth of glioma cell lines or other tumor cell lines in vivo as a result of inhibited tumor angiogenesis.
In addition, hypoxia, a large number of growth factors and cytokines, e.g. Epidermal Growth Factor, Transforming Growth Factor a, Transforming Growth Factor A, Interleukin 1, and Interleukin 6, induce the expression of VEGF in cell experiments. Angiogenesis is regarded as an absolute prerequisite for those tumors that grow beyond a maximum diameter of about 1-2 mm; up to this limit, oxygen and nutrients may be supplied to the tumor cells by diffusion. Every tumor, regardless of its origin and its cause, is thus dependent on angiogenesis for its growth after it has reached a certain size.
Three principal mechanisms play an important part in the activity of angiogenesis inhibitors against tumors: 1) inhibition of the growth of vessels, especially capillaries, into avascular resting tumors, with the result that there is no net tumor growth owing to the balance that is achieved between apoptosis and proliferation; 2) prevention of the migration of tumor cells owing to the absence of bloodflow to and from tumors; and 3) inhibition of endothelial cell proliferation, thus avoiding the paracrine growth-stimulating effect exerted on the surrounding tissue by the endothelial cells that normally line the vessels.
Inhibitors of cyclin-dependent kinases, e.g., Alvocidib (U.S. Pat. No. 4,900,727; also known as flavopiridol) have been identified as a potentially useful therapeutic agents for a variety of cancers, including gastrointestinal and colon tumors, leukemias and myelomas (see, for example, Intl. J. Oncol., 1996, 9, 1143).
Inhibitors of tyrosine kinases, including Bcr-Abl, e.g., Gleevec, are useful for the treatment of chronic myeloid leukemia (CML), and potentially for treatment of other cancers that express these kinases, including acute lymphocytic leukemia (ALL) and certain solid tumors. Gleevec was approved for the treatment of inoperable and/or metastatic malignant gastrointestinal stromal tumors (GISTs).
Inhibitors of Flt3 tyrosine kinase, e.g., CEP-701 (U.S. Pat. No. 4,923,986) and Midostaurin (U.S. Pat. No. 5,093,330), have potential utility for the treatment of a variety of cancers (Cancer Res., 1999, 59, 10).
Inhibitors of MAP Erk kinase, e.g., PD-184352 (U.S. Pat. No. 6,251,943), have been identified as potentially useful therapeutic agents for a variety of oncological disorders, including colon, breast, pancreatic and non-small-cell lung cancers (see, for example, Proc. Am. Soc. Clin. Oncol., 2003, 22, abstract 816).
Other kinase inhibitors, e.g., doramapimod (U.S. Pat. No. 6,319,921), have been identified as potentially useful therapeutic agents for the treatment of inflammatory diseases such as rheumatoid arthritis, psoriasis and Crohn's disease.
Other kinase inhibitors, e.g., BAY-43-9006 (U.S. Publication No. 2002/0165394) have been identified as potentially useful therapeutic agents for a variety of cancers including gastrointestinal and colon tumors, leukemia and carcinoma (Curr. Pharm. Design, 2002, 8, 2269).
Cytokine receptors are critical for the development and homeostasis of immune cells. These receptors all require the cytoplasmic tyrosine kinase JAK3 for signaling (Changelian, P. S. et al., Science, 2003, 302, 875). CP-690,550 (WO 02,096,909) is an orally available Janus kinase (JAK)-3 inhibitor, for the potential treatment of transplant rejection and psoriasis.
Thus, there is a need for therapeutic agents that are kinase inhibitors with improved pharmacological properties, e.g., drugs having improved kinase-inhibitory activity and pharmacokinetic properties, including improved oral bioavailability, greater potency and extended effective half-life in vivo. Such inhibitors would have therapeutic potential as, e.g., anticancer agents. Such kinase inhibitory compounds may be used to treat breast cancer, non-small cell lung cancers (NSCLCs), adenocarcinomas, squamous cell cancer of the lung, oesophageal cancer, gastrointestinal cancer, colon cancer, rectal cancer, stomach cancer, prostate cancer, leukaemia, ovarian cancer, bronchial cancer, pancreatic cancer, thyroid cancer, uterine cancer, brain cancer, lung squamous cell cancer, bladder cancer, gastric cancer, head and neck cancer, gynaecological and thyroid tumors, to prevent the development of resistance (multi-drug resistance) in cancer treatment with other chemotherapeutic drugs or remove existing resistance to other chemotherapeutic drugs, retinopathies, hemangioblastoma, hemangioma, and neoplastic diseases, gliomas, to inhibit tumor angiogenesis, myelomas, chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), inoperable and/or metastatic malignant gastrointestinal stromal tumors (GISTs), treatment of inflammatory diseases such as rheumatoid arthritis, Crohn's disease, treatment of cell proliferation diseases, and for the treatment of transplant rejection and psoriasis.
New kinase inhibitors should have fewer side effects, less complicated dosing schedules, and be orally active. In particular, there is a need for a less onerous dosage regimen, such as one pill, once per day.
Assay methods capable of determining the presence, absence or amounts of kinase inhibition are of practical utility in the search for inhibitors as well as for diagnosing the presence of conditions associated with kinase activity.